The overall goal of this project continues to be to understand the molecular mechanism of budding of transport vesicles, with special reference to the Golgi. With the support of this grant, coated transport vesicles budding from Golgi membranes (COPI vesicles) and a general principle for budding (utilizing coatomer and the GTPase ARF) were uncovered. We now seek deeper insights into budding mechanisms and the role of transport vesicles in cell physiology by addressing new, thematically-related questions concerning: 1) how the process of budding is coupled to packaging of essential membrane proteins, such as v-SNAREs and certain cargo receptors, mainly utilizing the recently achieved reconstitution of budding from synthetic lipid bilayers; 2) how distinct species of anterograde and retrograde-selective COPI vesicles can bud from the same Golgi membranes, utilizing the above approach and immuno-electron microscopy; 3) how transport signal peptides interact with their receptor, the gamma-COP subunit of coatomer, isolating domains by limited proteolysis and utilizing X-ray crystallography; and 4) how recently discovered Rim-Derived (RIDE)-vesicles bud off from Golgi stacks enclosing aggregates that are far too large for standard COPI vesicles, utilizing mainly cell-free budding reactions, protein purification, and immuno-electron microscopy. The process of vesicular transport is of basic importance to biology and medicine, underlying the compartmental organization of the cytoplasm and the pathways of exocrine, endocrine, and neurosecretion. Knowledge of the mechanism of action of gene products in these pathways, and their allelic variants, will likely be important for predictive medicine and impact upon management of common diseases such as cancer, diabetes, cardiovascular disease and diseases of the central nervous system.